scholarly journals Purification and properties of 5-aminolaevulinate dehydratase from human erythrocytes

1985 ◽  
Vol 230 (1) ◽  
pp. 25-34 ◽  
Author(s):  
P N B Gibbs ◽  
A G Chaudhry ◽  
P M Jordan
Keyword(s):  
Enzyme Activity ◽  
Dissociation Constant ◽  
Specific Activity ◽  
Enzymic Activity ◽  
Thiol Groups ◽  
General Application ◽  
Erythrocyte Enzymes ◽  
Human Enzyme ◽  
Purification And Properties ◽  
Porphobilinogen Synthase

A new procedure for the isolation of homogeneous human 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) is described in which the enzyme is purified 35000-fold and in 65-74% yield. The specific activity of the purified enzyme, 24 units/mg, is the highest yet reported. An efficient stage for the removal of haemoglobin is incorporated in the method, which has general application to the purification of other erythrocyte enzymes. The erythrocyte dehydratase (Mr 285 000) is made up of eight apparently identical subunits of Mr 35 000. The enzyme is sensitive to oxygen, and its activity is maintained by the presence of thiols such as dithioerythritol. Zn2+ is obligatory for enzyme activity, the apoenzyme being essentially inactive (approximately equal to 12% of control) when assayed in buffers devoid of Zn2+. Addition of Zn2+ to the apoenzyme restores activity as long as the sensitive thiol groups are fully reduced; optimal stimulation occurs between 100 and 300 microM-Zn2+. The human enzyme is inhibited by Pb2+ in a non-competitive fashion [KiI (dissociation constant for E X S X Pb2+ complex) = 25.3 +/- 3.0 microM; KiS (dissociation constant for E X Pb2+ complex) = 9.0 +/- 2.0 microM]. Modification of thiol groups, inactivation by oxidation, alkylation or reaction with thiophilic reagents demonstrates the importance of sensitive thiol groups for full enzymic activity.

scholarly journals Purification and Properties of a Glucuronan Lyase from Sinorhizobium meliloti M5N1CS (NCIMB 40472)

2001 ◽  
Vol 67 (11) ◽  
pp. 5197-5203 ◽  
Author(s):  
Alexandre Da Costa ◽  
Philippe Michaud ◽  
Emmanuel Petit ◽  
Alain Heyraud ◽  
Philippe Colin-Morel ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Sinorhizobium Meliloti ◽  
Specific Activity ◽  
Protein Sequences ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Terminal Amino Acid ◽  
Purification And Properties ◽  
Terminal Amino ◽  
Terminal Amino Acid Sequence

ABSTRACT A glucuronan lyase extracted from Sinorhizobium meliloti strain M5N1CS was purified to homogeneity by anion-exchange chromatography. The purified enzyme corresponds to a monomer with a molecular mass of 20 kDa and a pI of 4.9. A specific activity was found only for polyglucuronates leading to the production of 4,5-unsaturated oligoglucuronates. The enzyme activity was optimal at pH 6.5 and 50°C. Zn2+, Cu2+, and Hg2+ (1 mM) inhibited the enzyme activity. No homology of the enzyme N-terminal amino acid sequence was found with any of the previously published protein sequences. This enzyme purified fromS. meliloti strain M5N1CS corresponding to a new lyase was classified as an endopolyglucuronate lyase.

scholarly journals The reaction of aldolase with 2-methylmaleic anhydride

1970 ◽  
Vol 116 (5) ◽  
pp. 843-849 ◽  
Author(s):  
I. Gibbons ◽  
R. N. Perham
Keyword(s):  
Enzyme Activity ◽  
Maleic Anhydride ◽  
Enzymic Activity ◽  
Side Reactions ◽  
Rabbit Muscle ◽  
Thiol Groups ◽  
Amino Groups ◽  
Partial Loss ◽  
Reaction Proceeds ◽  
Citraconic Anhydride

1. The reaction of rabbit muscle aldolase with 2-methylmaleic anhydride is described. All the protein amino groups can be reversibly blocked. 2. As the reaction proceeds, the enzyme activity decreases until, at about 50% citraconylation of amino groups, the enzyme is completely inhibited. At this stage, little or no dissociation of the enzyme tetramer is observed and 75% of the activity is recoverable on unblocking the amino groups. 3. At 80% blocking, the enzyme is completely dissociated but little enzymic activity is recoverable after unblocking. Inability to recover activity after citraconylation and unblocking correlates with the onset of dissociation of the citraconyl-aldolase seen on ultracentrifugation. 4. The only irreversible modification of the enzyme primary structure detectable after the citraconylation and unblocking reactions is the partial loss of thiol groups. It is probable that this is responsible for the inability to reform active enzyme from the citraconylated subunit. 5. Other reversible side reactions of maleic anhydride and citraconic anhydride that may occur with proteins are discussed.

scholarly journals PREPARATION, PURIFICATION AND PROPERTIES OF LIPASE FROM HEPATOPANCREAS OF TRA (PANGASIUS) CATFISH

2011 ◽  
Vol 14 (3) ◽  
pp. 5-11
Author(s):  
Thy Bao Vuong ◽  
Lam Bich Tran ◽  
Duan Luu
Keyword(s):  
Heavy Metals ◽  
Molecular Weight ◽  
Enzyme Activity ◽  
Crude Extract ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Purification And Properties ◽  
Temperature Optima

Lipase from the hepatopancreas of Tra (Pangasius) catfish was purified by ammonium sulfate fractionation, followed by ion-exhange chromatography on DEAE Cellulose and gel filtration Sephadex G-75. The preparation was homogeneous on polyacrylamide disc gel electrophoresis. The specific activity of the purified enzyme was 37.95 times higher than that of the crude extract. The enzyme showed a molecular weight of 57000 Da. The pH and temperature optima of purified lipase were 8 and 500C respectively. Enzyme activity was enhanced by Ca2+ but inhibited by heavy metals Zn2+, Cd2+, Mg2+.

scholarly journals Purification and properties of adenosine triphosphate–creatine phosphotransferase from muscle of the dogfish Scylliorhinus canicula

1972 ◽  
Vol 128 (5) ◽  
pp. 1241-1253 ◽  
Author(s):  
B. Simonarson ◽  
D. C. Watts
Keyword(s):  
Creatine Kinase ◽  
Specific Activity ◽  
Thiol Group ◽  
Effect Of Temperature ◽  
Thiol Groups ◽  
High Concentration ◽  
Nitrobenzoic Acid ◽  
Purification And Properties ◽  
Michaelis Constants

1. Creatine kinase occurs in high concentration in the soluble proteins of dogfish muscle. A fourfold purification gives essentially pure enzyme but with a low specific activity. This appears to be a property of the native enzyme and not a result of the isolation procedures used. 2. The amino acid composition is similar to that of other phosphagen kinases, but the enzyme differs from mammalian creatine kinases in having four thiol groups readily reactive towards 5,5′-dithiobis-(2-nitrobenzoic acid). Titration of two thiol groups is accompanied by almost complete loss of activity. The remaining two thiol groups react at different rates, suggesting that modifying the third thiol group affects the reactivity of the fourth thiol group. 3. The enzyme is markedly protected against inactivation by iodoacetamide by MgATP or MgADP. Addition of creatine to MgADP decreases protection, but the further addition of Cl− restores protection to the original value. The quaternary MgADP–creatine–enzyme–nitrate complex protects very strongly as is found for the rabbit enzyme. The involvement of the conformational state of the enzyme in such effects is discussed. 4. Creatine kinase from both dogfish and rabbit is equally sensitive to urea denaturation. Urea protects the dogfish enzyme by about 9% against inhibition by iodoacetamide. 5. The formation of a hybrid between the dogfish and rabbit enzymes in vitro has been demonstrated. 6. At high substrate concentrations the dogfish enzyme shows apparent ordered kinetics. The effect of temperature on Vmax. and the Michaelis constants for MgATP and creatine were determined. These and changes in the apparent activation energy suggest that limited adaptation has occurred commensurate with physiological need.

scholarly journals Purification and properties of rabbit spermatozoal acrosomal neuraminidase

1977 ◽  
Vol 161 (2) ◽  
pp. 193-200 ◽  
Author(s):  
P N Srivastava ◽  
H Abou-Issa
Keyword(s):  
Enzyme Activity ◽  
Sialic Acid ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Submaxillary Gland ◽  
Subsequent Treatment ◽  
Major Band ◽  
Acrosomal Membrane ◽  
Purification And Properties ◽  
Freeze Dried

Treatment of rabbit spermatozoa with 50mM-MgCl2 removes the plasma and the outer acrosomal membranes. Subsequent treatment with the detergents Hyamine 2389 and Triton X-100 solubilizes spermatozoal neuraminidase bound to the inner acrosomal membrane. The enzyme was further purified by DEAE-cellulose, Sephadex G-150 and Bio-Gel P-300 column chromato. The enzyme showed a single major band, with the possibility of some minor contaminants, on disc-gel electrophoresis. It had a specific activity of 0.37 micronmal of sialic acid released/min per mg with purified boar Cowper's-gland mucin as the substrate. The enzyme had marked specificity for 2 leads to 6′-linked sialic acid in glycoproteins. The Km of spermatozoal neuraminidase was 1.72 X 10(-6)M with Cowper's-gland mucin, 1.17 X 10(-5)M with fetuin and 8.8 X 10(-4)M with sialyl-lactose as a substrates. The Vmax. was 0.112 micronmol/min per mg with the Cowper's-gland mucin, 0.071 micronmol/min per mg with fetuin and 0.033 micronmol/min per mg with sialyl-lactose as substrate. The enzyme hydrolysed sheep submaxillary-gland mucin as readily as the Cowper's-gland mucin. The optimum of enzyme activity was at pH 5.0 on the Cowper's-gland mucin and at pH4.3 on sialyl-lactose. The enzyme activity was unaffected by 20mM-Na+ and-K+, but was inhibited by 20mM-Ca2+,-Mn2+,-Co2+ and -Cu2+. The enzyme was unstable in dilute solutions, but could be stored indefinitely freeze-dried at --20 degrees C.

Purification of Glycerol Kinase by "Dye-Ligand" Chromatography and Hydrophobic Interaction Chromatography on Bead-Cellulose Derivatives

1993 ◽  
Vol 58 (2) ◽  
pp. 445-451 ◽  
Author(s):  
Vladimír Žúbor ◽  
Albert Breier ◽  
Marta Horváthová ◽  
Dagmar Hagarová ◽  
Peter Gemeiner ◽  
...  
Keyword(s):  
Hydrophobic Interaction ◽  
Specific Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Glycerol Kinase ◽  
Enzymic Activity ◽  
Cellulose Derivatives ◽  
Long Term Storage ◽  
Bead Cellulose ◽  
Term Storage

The crude extract of cytosole enzymes was obtained from homogenized cells of Saccharomyces cerevisiae by partition. The enzyme was then isolated from the lower aqueous phase displaying higher glycerol kinase activity by dye-ligand chromatography on Cibacron Blue (CB) or Remazol Brilliant Blue R (RB)-derivatized bead-cellulose, ATP being the eluent. The specific activity of glycerol kinase rised more than 10 and 7-times after affinity dye-ligand chromatography and hydrophobic interaction chromatography, respectively. Glycerol kinase obtained by the latter method was purified by CB-bead cellulose. The final preparation maintained its enzymic activity without noticeable losses during a long-term storage at 4 °C in dark.

scholarly journals Mechanism of porphobilinogen synthase. Requirement of Zn2+ for enzyme activity.

1980 ◽  
Vol 255 (5) ◽  
pp. 2030-2035
Author(s):  
D.R. Bevan ◽  
P. Bodlaender ◽  
D. Shemin
Keyword(s):  
Enzyme Activity ◽  
Porphobilinogen Synthase

scholarly journals Mechanism of porphobilinogen synthase. Possible role of essential thiol groups.

1977 ◽  
Vol 252 (24) ◽  
pp. 8965-8974 ◽  
Author(s):  
G.F. Barnard ◽  
R. Itoh ◽  
L.H. Hohberger ◽  
D. Shemin
Keyword(s):  
Thiol Groups ◽  
Porphobilinogen Synthase

scholarly journals Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69

Catalysts ◽  
2018 ◽  
Vol 8 (8) ◽  
pp. 325 ◽  
Author(s):  
He Chen ◽  
Jie Huang ◽  
Binyun Cao ◽  
Li Chen ◽  
Na Song ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Lactobacillus Plantarum ◽  
Industrial Development ◽  
Extraction Efficiency ◽  
Specific Activity ◽  
Cell Envelope ◽  
Serine Proteinase ◽  
Kinetic Studies ◽  
Predicted Values ◽  
Hydrolyze Whey Protein

Lactobacilli cell-envelope proteinases (CEPs) have been widely used in the development of new streams of blockbuster nutraceuticals because of numerous biopharmaceutical potentials; thus, the development of viable methods for CEP extraction and the improvement of extraction efficiency will promote their full-scale application. In this study, CEP from a novel wild Lactobacillus plantarum LP69 was released from cells by incubating in calcium-free buffer. The extraction conditions of CEP were optimized by response surface methodology with the enzyme activity and specific activity as the detective marker. The optimal extraction conditions were: time of 80 min, temperature of 39 °C and buffer pH of 6.5. Under these conditions, enzyme activity and specific activity were (23.94 ± 0.86) U/mL and (1.37 ± 0.03) U/mg, respectively, which were well matched with the predicted values (22.12 U/mL and 1.36 U/mg). Optimal activity of the crude CEP occurred at pH 8.0 and 40 °C. It is a metallopeptidase, activated by Ca2+, inhibited by Zn2+ and ethylene-diamine-tetra-acetic acid, and a serine proteinase which is inhibited by phenylmethylsulfonyl fluoride. Kinetic studies showed that CEP from LP69 could hydrolyze whey protein, lactoglobulin and casein. Our study improves the extraction efficiency of CEPs from LP69, providing the reference for their industrial development.

Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves

2005 ◽  
Vol 32 (9) ◽  
pp. 839
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Heat Treatment ◽  
Thermal Stability ◽  
Enzyme Activity ◽  
Inorganic Phosphate ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg–1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

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